Discovery of thiophene-2-carboxylic acids as potent inhibitors of HCV NS5B polymerase and HCV subgenomic RNA replication. Part 1: Sulfonamides

Article abstract of DOI:10.1016/j.bmcl.2003.10.067

The discovery of a novel class of HCV NS5B polymerase inhibitors, 3-arylsulfonylamino-5-phenyl-thiophene-2-carboxylic acids is described. SAR studies have yielded several potent inhibitors of HCV polymerase as well as of HCV subgenomic RNA replication in

Full text of DOI:10.1016/j.bmcl.2003.10.067

Bioorganic & Medicinal Chemistry Letters 14 (2004) 793–796
Discovery of thiophene-2-carboxylic acids as potent inhibitors
of HCV NS5B polymerase and HCV subgenomic RNA
replication. Part 1: Sulfonamides
Laval Chan,* Sanjoy K. Das, T. Jagadeeswar Reddy, Carl Poisson, Melanie Proulx,
Oswy Pereira, Marc Courchesne, Caroline Roy, Wuyi Wang, Arshad Siddiqui,
Constantin G. Yannopoulos, Nghe Nguyen-Ba, Denis Labrecque, Richard Bethell,
Martine Hamel, Philippe Courtemanche-Asselin, Lucille L’Heureux, Maud David,
Olivier Nicolas, Stephanie Brunette, Darius Bilimoria and Jean Bedard
Shire BioChem Inc., 275 Armand-Frappier, Laval, Quebec, Canada H7V 4A7
Received 19 August 2003; accepted 31 October 2003
Abstract—The discovery of a novel class of HCV NS5B polymerase inhibitors, 3-arylsulfonylamino-5-phenyl-thiophene-2-car-
boxylic acids is described. SAR studies have yielded several potent inhibitors of HCV polymerase as well as of HCV subgenomic
RNA replication in Huh-7 cells.
# 2003 Elsevier Ltd. All rights reserved.
In 1989, Hepatitis C virus (HCV) was identified as the
pathogen responsible for the majority of the cases of
non-A non-B hepatitis. This positive strand RNA virus
of the Flaviviridae family chronically infects approxi-
mately 3% of the world population or 175 million indi-
viduals causing severe liver malfunction and morbidity.
To date, there is no prophylactic vaccine and the
recommended treatments, interferon a2b or a pegylated
conjugate (PEG-Intron1) alone or in combination with
ribavirin provide sustained viral suppression only in a
fraction of the cases. Furthermore, severe side effects
often limit compliance to the therapy. As a result,
intensive efforts have been focused on the development
of an efficacious and well-tolerated low molecular
weight anti-HCV agent. The major obstacle towards
this goal has been the lack of a cell culture system and/
or a convenient small animal model to propagate the
virus.1 A major breakthrough was recently made by
Bartenschlager and co-workers who engineered a Huh-7
human hepatoma cell line that supports replication of
subgenomic HCV RNA replicons (replicon cell line).2
These replicons encode all the non-structural proteins
(NS3, NS4A, NS4B, NS5A and NS5B) of HCV and
thus provide a useful surrogate antiviral model for
evaluating compounds which show activity against any
of these functional targets of HCV.
The HCV NS5B RNA dependent RNA polymerase3 is
one of the most studied target because it has been
shown that a functional polymerase is essential for
infectivity in chimpanzees.4 Recently, a group from
GlaxoSmithKline Pharmaceuticals reported the anti
HCV NS5B polymerase activity of several benzo-1,2,4-
thiadiazine analogues which also inhibited the replica-
tion of HCV replicons.5 An inhibitor from Japan
Tobacco Inc. is also reported to be undergoing Phase II
clinical trials.6 We have recently described the identifi-
cation and SAR studies of a novel class of HCV poly-
merase inhibitors which are characterized by
a
N,N-disubstituted phenylalanine moiety A.7 Crystal
structures of inhibitor–NS5B HCV polymerase com-
plexes were also generated and this led to the identifi-
cation of an allosteric binding site located about 35 A
from the active site.8
Compound libraries were screened against HCV NS5B
polymerase using the assay described previously;7 selec-
ted compounds were then screened in a HCV replicon
* Corresponding author: Tel.: +1-450-978-7802; fax: +1-450-978-
7777; e-mail: lchan@ca.shire.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.067

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L. Chan et al. / Bioorg. Med. Chem. Lett. 14(2004) 793–796
assay using a Huh-7 derived cell line (5.2 cells) which
carries a replicon containing the luciferase reporter
gene.9 RNA replication can thus be conveniently mea-
sured by determining the level of luciferase activity. The
concentration of compound required to suppress RNA
replication by 50% was expressed as IC50. Since cyto-
toxic compounds are detrimental to cell viability and
hence HCV RNA replication, cytotoxicity was deter-
Scheme 2. Preparation of 10–12. Reagents: (a) LDA, THF, À78 ꢀC
then (CF2Br)2, 92%; (b) (i) arylboronic acid, Pd(PPh3)4, toluene/
MeOH, Na2CO3 (aq), 65 ꢀC; (ii) TFA/CH2Cl2 (1:1), rt; (iii) LiOH,
THF, MeOH, H2O(3:2:1), rt, then HCl (1 N); (iv) Method B.
3
mined by measuring the incorporation of H-thymidine
in Huh-7 cells. The concentration required to suppress
3H-thymidine uptake by 50% was expressed as CC50
and the Selectivity Index (SI) was calculated as the ratio
of CC50 over IC50. Our screening efforts led to the
identification of a novel class of low molecular weight
inhibitors based on a 5-phenyl-thiophene scaffold 1
which bears a carboxamide and a sulfonamide at the 2-
and 3-positions respectively.
presence of a methyl ester without the added compli-
cation of cross-condensation from the anion. The
adjacent bulky BOC protecting group probably steri-
cally hinders the methyl ester carbonyl from nucleo-
philic attack. Regioisomer 13 was prepared from the
corresponding commercially available 2-amino thio-
phene derivative. The synthesis of regioisomer 14 is
depicted in Scheme 3. The 4-amino thiophene deriva-
tive 22 used in the preparation of regioisomer 14 was
obtained from nitration12 of 5-bromothiophene 21 fol-
lowed by Suzuki coupling with phenylboronic acid and
reduction with tin(II) chloride. In contrast direct nitra-
tion of commercial 5-phenyl-2-methyl carboxylate
resulted in exclusive nitration of the phenyl ring. Urea
17 and carbamate 18 were prepared from the corre-
sponding isocyanate and chloroformate, respectively.
Preliminary SAR data showed the 2-carboxy analogue 2
to be 3-fold more potent than the corresponding car-
boxamide 1. Furthermore, both the methyl carboxylate
and 5-desphenyl derivatives of 2 were inactive against
NS5B polymerase. It is interesting to note that in our
previously described series, a carboxylic acid moiety was
essential for polymerase inhibition.7 Taking these find-
ings into consideration, a systematic modification of the
3- and 5-positions was undertaken. In addition, the
importance of the relative position of the three func-
tionalities on the thiophene nucleus was also studied.
The compounds described in this study were evaluated
for inhibition of HCV NS5B polymerase and selected
compounds were also evaluated for inhibition of HCV
subgenomic RNA replication in the replicon cell line. A
systematic study of the importance of the substituents
on the phenyl ring of the sulfonamide was undertaken
and the results are depicted in Table 1.
From the in vitro polymerase inhibition of compounds
4–7, it became apparent that single substitution at the
ortho, meta or para position does not provide any
potency enhancement compared to the trisubstituted
phenyl sulfonamide 2. No improvement was also
observed for the 2,5-dimethyl substituted analogue 3.
However, the 2,4-disubstituted analogues provided a
5-fold enhancement in potency; the 4-chloro-2-methyl,
analogue 8 and the 2,4-dimethyl derivative 9 had IC50’s
of 0.75 and 1 mM, respectively. Polymerase inhibition
was also enhanced by introduction of an acyl or methyl-
sulfonyl unit on the para position of the 5-phenyl ring.
The ortho methyl substituted analogues 10 and 12 had an
IC50 of approximately 1.5 mM, whereas combining the
acyl unit with the 2,4-dimethyl derivative gave the most
potent compound (11, IC50 0.39 mM) of this study.
All the sulfonamides and amides described in this study
were prepared by the reaction of commercially available
acid or sulfonyl chlorides10 with a 3-amino thiophene
derivative in the presence of base (Scheme 1). Sub-
stituted 5-phenyl thiophene analogues 10–12 were
obtained under Suzuki coupling conditions from the
corresponding 5-bromothiophene 20 (Scheme 2). This
compound was conveniently prepared from LDA medi-
ated 5-proton abstraction from 19 followed by quench-
ing with 1,2-dibromotetrafluoroethane11 to give the
desired 5-bromo derivative 20 in 92% yield (Scheme 2).
This approach represents a highly efficient way of
effecting a lithium anion mediated bromination in the
Most of the compounds described in Table 1 were
deemed too toxic to be able to differentiate between
Scheme 1. Preparation of 2–9 and 13. Reagents: compounds 2, 6, 13:
Method A (R=Me) (i) sulfonyl chloride, pyridine; (ii) LiOH, THF/
MeOH/H2O(3:2:1), rt; then HCl (1 N). Compounds 3–5, 7–9: Method
B (R=H) sulfonyl chloride, Na2CO3 dioxane/H2O(1:1); then HCl
(1 N).
Scheme 3. Preparation of 14. Reagents: (a) (i) NO2BF4, CH3CN,
75%; (ii) PhB(OH)2, Pd(PPh3)4, toluene/MeOH, Na2CO3 (aq) 65 ꢀC,
.
75%; (iii) SnCl2 2H2O, AcOEt, 57%. (b) (i) LiOH, THF, MeOH, H2O
(3:2:1), rt, 93%, then HCl (1 N); (ii) Method B, 48%.

L. Chan et al. / Bioorg. Med. Chem. Lett. 14(2004) 793–796
Table 1. NS5B Polymerase and replicon inhibition16 Table 2. HCV polymerase activity of regiosomers16
795
R
Ar
HCV NS5B Replicon
Huh-7
polymerase IC50 (mM) CC50 (mM)
IC50 (mM)
Compd
Position
HCV NS5B polymerase
COOH
NHSO2Ar
Ph
IC50 (mM)
13
14
3
2
2
4
5
5
20
>50
1
14
2
H
H
H
H
H
H
H
H
5.4
14
11
80
24
Table 3. Replacement of sulfonyl with a carbonyl moiety16
3
13
4
5.2
7.4
4.6
4.1
0.75
1
24
76
5
100
33
112
79
Compd
X
R
HCV NS5B polymerase IC50 (mM)
15
16
17
18
—
—
NH
2-Me
4-Me
4-Cl
14
12
>50
6
O4-Cl
13
7
77
92
addition to viral RNA template, the polymerase
enzyme is likely associated with viral and cellular pro-
teins. A recent report has provided evidence for mod-
ulation of HCV polymerase activity by NS3 and
NS4B.13 In addition, the inability of these compounds
to achieve sufficient intra-cellular concentration for
inhibition cannot be ignored at this point.
8
13
54
9
5
57
10
11
1.4
0.39
1.8
12
147
100
>200
Regioisomers 13–14 were also less active (Table 2) than
the series of compounds depicted in Table 1. O ther
3-amino derivatives such as amide, urea and carbamate
(15–18) were less active (Table 3). The 2,3,5 arrangement
of substituents on the thiophene scaffold appears to be
necessary for activity but whether this requirement is rela-
ted to the angles of bisection remains to be determined.14
8
12 MeSO2
>100
In summary, a novel class of HCV NS5B polymerase
inhibitors has been identified and modification of the
lead has resulted in a series of potent polymerase inhi-
bitors and some of which also inhibited HCV sub-
genomic RNA replication. However, it is not clear at
this point why in some cases, in vitro inhibition of HCV
NS5B polymerase fails to translate into inhibition of
replicon RNA replication. Further studies in optimizing
this series of compounds are reported in the following
publication.15
anti-HCV activity and cytotoxicity; analogues 2–8 had
selectivity indices (SI) of less than 5. However, com-
pounds with 2,4-disubstituted phenylsulfonamides and/
or those with acyl moieties on 5-phenyl gave anti-HCV
replicon IC50’s of 5–13 mM with SI’s of about 12. The
lack of inhibition of HCV replication with methylsulfo-
nyl analogue 12 was surprising considering the level of
HCV polymerase inhibition (IC50 1.8 mM). This dis-
crepancy may be attributed to conformational differ-
ences that exist between the polymerase/homopolymeric
template/primer complex in the in vitro assay and the
cellular environment in the replicon assay where, in
Acknowledgements
We thank Therese Godbout for her assistance in the
preparation of this manuscript.

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L. Chan et al. / Bioorg. Med. Chem. Lett. 14(2004) 793–796
14.
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16. IC50’s values were determined singly from dose-response
curves using 11 concentrations for each compound.
Curves were fitted to data points using nonlinear regres-
sion analysis and IC50’s values were interpolated from the
resulting curves using GraphPad Prism software, version
2.0 (GraphPad Software, Inc., San Diego, CA, USA). A
positive control was included as an internal standard in
each set of experiments, and results were considered
accurate only when the IC50 value of the positive control
was within 0.45Æ0.16 mM. Similarly, IC50’s in replicons
were determined using 12 concentrations in duplicate and
data were considered accurate when IC50 of a positive
control was within 8.9Æ2.4 mM. CC50’s in Huh-7 cells
were determined from 5 concentrations in quadruplicate
and data were taken when CC50 of a positive control was
within 62Æ10 mM.